EP1700864A1 - Procédé pour la production de S-adénosylméthionine par fermentation - Google Patents

Procédé pour la production de S-adénosylméthionine par fermentation Download PDF

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Publication number
EP1700864A1
EP1700864A1 EP06003718A EP06003718A EP1700864A1 EP 1700864 A1 EP1700864 A1 EP 1700864A1 EP 06003718 A EP06003718 A EP 06003718A EP 06003718 A EP06003718 A EP 06003718A EP 1700864 A1 EP1700864 A1 EP 1700864A1
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Prior art keywords
sam
cmr
mdfa
gene
strain
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German (de)
English (en)
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Thomas Dr. Schlösser
Susanne Dr. Leonhartsberger
Renate Flinspach
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Wacker Chemie AG
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Consortium fuer Elektrochemische Industrie GmbH
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/38—Nucleosides
    • C12P19/40—Nucleosides having a condensed ring system containing a six-membered ring having two nitrogen atoms in the same ring, e.g. purine nucleosides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)

Definitions

  • the invention relates to a process for the fermentative production of S-adenosylmethionine.
  • SAM S-adenosylmethionine
  • the former object is solved by a microorganism strain of S-adenosylmethionine secreted, characterized in that it has an increased activity of cmr (mdfA) gene product.
  • the microorganism strain according to the invention can be prepared from an initial strain and has an increased activity of the cmr (mdfA) gene product compared with the starting strain.
  • a strain of microorganism preferably has an increased activity of the cmr (mdfA) gene product compared with the starting strain if a cell of the strain is at least a factor of 2, preferably about 1, compared to a cell of a wild-type microorganism strain with cmr (mdfA) gene product has at least the factor 5, increased activity of cmr (mdfA) gene product.
  • cmr (mdfA) gene product Experiments to determine the activity of the cmr (mdfA) gene product are described in the scientific literature (Edgar and Bibi, J. Bacteriol 179, 2274-2280).
  • the cmr (mdfA) gene of E. coli was identified as chloramphenicol export protein Cmr in 1996 (Nilsen et al., J. Bacteriol 178, 3188-3193). In 1997, the cmr (mdfA) gene was again described as a multidrug efflux protein MdfA with a broad substrate scope (Edgar and Bibi, J. Bacteriol 179, 2274-2280).
  • the Cmr (MdfA) protein belongs to the family of MF (S) [Major Facilliator (Superfamily)] transporters and transports both lipophilic, uncharged substrates such as. B.
  • Cmr (MdfA) protein has a broad substrate scope, it is surprising to those skilled in the art that Cmr (MdfA) can function as a SAM export protein since SAM has no structural similarities with the substrates described so far.
  • cmr (mdfA) and the amino acid sequence of the Cmr (MdfA) protein show no homology to the previously known SAM transport genes and SAM transport proteins from yeast and man. A prediction of whether a substance can act as a substrate for the Cmr (MdfA) protein is also not possible due to the largely unknown transport mechanism. Furthermore, it is completely surprising for a person skilled in the art that in the microorganism strain according to the invention with SAM, a strongly hydrophilic, positively charged and at the same time cell-like molecule is transported out of the cell.
  • the invention therefore also relates to the use of the cmr (mdfA) gene product as an export protein in the production of SAM.
  • cmr (mdfA) gene and the cmr (mdfA) gene product (Cmr (MdfA) protein) are represented by the sequences SEQ ID NO. 1 or SEQ ID no. 2 characterized.
  • cmr (mdfA) genes are also to be understood as meaning those genes which code for a protein with chloramphenicol export or multidrug efflux activity and are labeled with the BESTFIT algorithm (GCG Wisconsin Package, Genetics Computer Group (GCG)). Madison, Wisconsin) has a sequence identity of greater than 30% to SEQ ID no. 1 have. A sequence identity of greater than 50% to SEQ ID no. 1. Particularly preferred is a sequence identity of greater than 70% to SEQ ID no. 1.
  • proteins with chloramphenicol export or multidrug efflux activity and with a sequence identity are larger in size 15% to SEQ ID no. 2 Algorithm BESTFIT (GCG Wisconsin Package, Genetic Computing Group (GCG) Madison, Wisconsin) as Cmr (MdfA) proteins.
  • cmr (mdfA) genes are also allelic variants of the cmr (mdfA) gene, in particular functional variants which are obtained by deletion, insertion or substitution of nucleotides from the nucleotide sequence shown in SEQ ID no. 1 derived sequence, but the enzymatic activity of the respective gene product must at least be preserved.
  • Microorganisms of the invention can be produced by standard molecular biology techniques.
  • Suitable starting strains are in principle all microorganisms which have the biosynthetic pathway for SAM, are accessible to recombinant processes and can be cultivated by fermentation.
  • Such microorganisms may be fungi, yeasts or bacteria.
  • they are bacteria of the phylogenetic group of Eubacteria.
  • Particularly preferred are microorganisms of the family Enterobacteriaceae and in particular of the species Escherichia coli.
  • the increase in the activity of the cmr (mdfA) gene product in the microorganism according to the invention is achieved, for example, by an increased expression of the cmr (mdfA) gene.
  • the copy number of the cmr (mdfA) gene may be increased in a microorganism and / or the expression of the cmr (mdfA) gene may be increased by suitable promoters.
  • Enhanced expression is preferably understood to mean that the cmr (mdfA) gene is expressed at least twice as much as in the parent strain.
  • An increased copy number of the cmr (mdfA) gene is preferably to be understood as meaning that additionally at least one chromosomally and / or plasmid-coded copy of the cmr (mdfA) gene is used in comparison to the starting strain.
  • the cmr (mdfA) gene can be cloned into multiple copy number per cell plasmid vectors (e.g., pUC19, pBR322, pACYC184 for E. coli) and introduced into the microorganism.
  • the cmr (mdfA) gene can be integrated several times into the chromosome of a microorganism.
  • integration method the known systems with temperate bacteriophages, integrative plasmids or integration via homologous recombination can be used.
  • the natural promoter and operator region of the gene can serve as a control region for the expression of a plasmid-encoded cmr (mdfA) gene.
  • cmr (mdfA) gene may also be by other promoters.
  • Corresponding promoter systems such as, for example, the constitutive GAPDH promoter of the gapA gene in E. coli or the inducible lac, tac, trc, lambda, ara or tet promoters are known to the person skilled in the art.
  • Such constructs can be used in a conventional manner on plasmids or chromosomally.
  • increased expression can be achieved by translational start signals, such.
  • the ribosome binding site or the start codon of the gene are present in optimized sequence on the respective construct or that according to the codon usage rare codons are replaced by more common codons.
  • Microorganism strains with the modifications mentioned are preferred embodiments of the invention.
  • cmr (mdfA) gene in plasmid vectors is carried out, for example, by specific amplification by means of the polymerase chain reaction using specific primers which capture the complete cmr (mdfA) gene, and subsequent ligation with vector DNA. fragments.
  • plasmids are already used which already contain promoters for enhanced expression, for example the constitutive GAPDH promoter of the gapA gene of E. coli.
  • vectors which already contain a gene / allele whose use leads to an increased biosynthesis of SAM such as, for example, a rat liver SAM synthetase (RLSS) allele, the metK gene (described in EP 0 647 712 A1 and EP 1 457 569 A1) or combinations of several SAM synthetases.
  • RLSS rat liver SAM synthetase
  • metK gene described in EP 0 647 712 A1 and EP 1 457 569 A1
  • plasmids containing multiple copies of a SAM synthetase gene can also be used.
  • Such vectors allow the direct production of microorganism strains according to the invention with high SAM overproduction from any microorganism strain.
  • the invention thus also relates to a plasmid which is characterized in that it contains a SAM synthetase gene and a cmr (mdfA) gene under the control of a promoter.
  • a plasmid may also contain combinations of SAM synthetase genes of different organisms or multiple copies of a SAM synthetase gene.
  • the cmr (mdfA) -containing plasmids are introduced into microorganisms and selected for example by means of antibiotic resistance to plasmid-carrying clones.
  • the invention thus also relates to processes for the preparation of a microorganism strain according to the invention, characterized in that an inventive plasmid is introduced into an initial strain.
  • the production of SAM with the aid of a microorganism strain according to the invention is carried out in a fermenter according to known methods.
  • the invention thus also relates to a process for the preparation of SAM, which is characterized in that a microorganism strain according to the invention is fermented in a fermentation medium and SAM is secreted into the fermentation medium.
  • a microorganism strain according to the invention is fermented in a fermentation medium and SAM is secreted into the fermentation medium.
  • the produced SAM is separated from the fermentation batch.
  • the cultivation of the microorganism strain in the fermenter takes place as a continuous culture, as a batch culture or preferably as a fed-batch culture.
  • a carbon source is metered in continuously during the fermentation.
  • the carbon source is preferably sugar, sugar alcohols or organic acids.
  • Glucose, lactose or glycerol are particularly preferably used as carbon sources in the process according to the invention.
  • the dosage of the carbon source is in a form which ensures that the content of carbon source in the fermenter is maintained within the range of 0.1-50 g / l during the fermentation. Particularly preferred is a range of 0.5-10 g / l.
  • the nitrogen source used in the process according to the invention is preferably ammonia, ammonium salts or protein hydrolyzates.
  • ammonia is used as a correction agent for pH stabilization, this nitrogen source is regularly replenished during the fermentation.
  • salts of the elements phosphorus, chlorine, sodium, magnesium, nitrogen, potassium, calcium, iron and in traces (i.e., in ⁇ M concentrations) salts of the elements molybdenum, boron, cobalt, manganese, zinc and nickel may be added.
  • organic acids eg, acetate, citrate
  • amino acids eg, isoleucine
  • vitamins eg, B 1 , B 12
  • yeast extract corn steep liquor, soybean meal or malt extract are used.
  • L-methionine or D / L-methionine may be added to the medium as a specific precursor for SAM synthesis in a concentration between 0.05 and 25 g / l.
  • the medium L-methionine or D, L-methionine is added continuously during the cultivation. Preference is given to a continuous addition of L-methionine or D / L-methionine between 0.05 g and 10 g per hour. Particularly preferred is a continuous addition of L-methionine or D / L-methionine between 0.1 g and 2 g per hour.
  • the incubation temperature for mesophilic microorganisms is preferably 15-45 ° C, more preferably 30-37 ° C.
  • the fermentation is preferably carried out under aerobic growth conditions.
  • the oxygen entry into the fermenter takes place with compressed air or with pure oxygen.
  • the pH of the fermentation medium during the fermentation is preferably in the pH range from 5.0 to 8.5, particularly preferred is a pH of 7.0.
  • the incubation of the strain is preferably carried out under aerobic cultivation conditions over a period of 16-150 h and in the region of the optimum growth temperature for the respective strain. Particularly preferred are cultivation times between 20 and 48 h.
  • the separation of SAM from the culture medium can be carried out by methods known to those skilled in the art, such as centrifugation of the medium to separate the cells, crossflow filtration to separate proteins and subsequent chromatographic purification, concentration, formulation or complexing of the product.
  • the detection and quantification of the SAM produced in the process according to the invention is carried out, for example, by means of chromatography (for example HPLC).
  • the plasmid pKP504 was linearized using the restriction endonuclease SspI .
  • the preparation of the plasmid pKP504 is described in patent EP-A-1 457 569, Example 4. This was followed by dephosphorylation of the linearized plasmid using an alkaline phosphatase (Roche, Mannheim). Finally, prior to ligation, purification of the linearized vector was performed using a QIAquick Nucleotide Removal Kit (Qiagen, Hilden) according to the manufacturer's instructions.
  • the GAPDH promoter-RLSS fragment was isolated from the SAM production plasmid pMSRLSSk (EP 1 457 569 A1) using the restriction endonucleases Ecl136II and StuI. After purification of the fragment by means of agarose gel electrophoresis with subsequent gel extraction (QIAquick Gel Extraction Kit, Qiagen, Hilden), a ligation of the GAPDH promoter-RLSS fragment and the Ssp I-linearized vector pKP504 was carried out by means of a T4 DNA ligase (Roche, Mannheim) according to manufacturer's instructions. The transformation of E.
  • coli cells of strain DH5 ⁇ (Invitrogen, Düsseldorf) with the ligation mixture was carried out by electroporation in a manner known to those skilled in the art.
  • the transformation mixture was applied to LB tetracycline agar plates (10 g / l tryptone, 5 g / l yeast extract, 5 g / l NaCl, 15 g / l agar, 20 mg / l tetracycline) and incubated overnight at 37 ° C.
  • the desired transformants were identified after plasmid isolation using a QIAprep Spin Miniprep Kit (Qiagen, Hilden) by restriction analysis.
  • plasmid pFL242 ( Figure 1), it is now possible to clone a further gene under the control of the constitutive GAPDH promoter of the E. coli GAPA gene.
  • the plasmid pFL242 which was used for the execution of the examples, was deposited on 17.02.2005 with the DSMZ (German collection for microorganisms and cell cultures GmbH, D-38142 Braunschweig) under the number DSM 17142 under Budapest contract.
  • the cmr (mdfA) gene from E. coli was amplified by polymerase chain reaction (PCR) using Taq DNA polymerase according to standard practice known to those skilled in the art.
  • the template used was the chromosomal DNA of the E. coli wild-type strain W3110 (ATCC 27325).
  • the primers used were the oligonucleotides cmr for (SEQ ID No: 3) with the sequence and cmr rev (SEQ ID No: 4) with the sequence used.
  • the DNA fragment obtained in the PCR with a length of about 1.3 kb was then purified by means of a DNA Adsorptionssklalchens the QIAprep Spin Miniprep Kit (Qiagen, Hilden) according to the manufacturer.
  • Two restriction endonuclease sites StuI and PacI were introduced into the PCR fragment via the primers cmr for and cmr rev.
  • the purified PCR fragment was cut with the restriction endonucleases Stu I (Roche, Mannheim) and PacI (New England Biolabs, Frankfurt am Main) under the conditions specified by the manufacturer, separated on an agarose gel and then purified using the QIAquick Gel Extraction Kit (Qiagen, Hilden) isolated from the agarose gel according to the manufacturer's instructions.
  • the vector pFL242 was cut with the restriction enzymes StuI and PacI under the conditions specified by the manufacturer.
  • the plasmid was then dephosphorylated by treatment with alkaline phosphatase (Roche, Mannheim) at the 5 'ends and then purified like the PCR fragment using QIAquick Gel Extraction Kit (Qiagen, Hilden).
  • the ligation of the PCR fragment with the cut and dephosphorylated vector was carried out according to the manufacturer's instructions using the T4 DNA ligase (Roche, Mannheim). Transformation of E. coli cells of strain W3110 (ATCC 27325) with the ligation mixture was performed by electroporation in a manner known to those skilled in the art.
  • the transformation mixture was applied to LB tetracycline agar plates (10 g / l tryptone, 5 g / l yeast extract, 5 g / l NaCl, 15 g / l agar, 20 mg / l tetracycline) and incubated overnight at 37 ° C.
  • the desired transformants were identified after a plasmid isolation using a QIAprep Spin Miniprep Kit (Qiagen, Hilden) by a restriction analysis and confirmed the absence of errors by sequence analysis.
  • plasmid pFL274 Figure 2 thus obtained, the cmr (mdfA) gene is under the control of the GAPDH promoter.
  • the plasmid pFL274 described in Example 2 was used by CaCl 2 method for the transformation of E. coli strain W3110 (ATCC 27325) and after selection on LB agar plates with 20 mg / l tetracycline, the plasmid was reisolated from one of the transformants, with Restriction endonucleases cleaved and checked. This strain is called W3110 / pFL274 and is suitable for the production of SAM.
  • strain W3110 / pFL274 was used for the fermentative production of SAM.
  • W3110 / pMSRLSSk was prepared analogously to Example 3 from W3110 and the plasmid pMSRLSSk.
  • the following medium was used for the culture: for 1 l of medium: CaCl 2 ⁇ 2 H 2 O 0.0147 g, MgSO 4 ⁇ 7 H 2 O 0.3 g, Na 2 MoO 4 ⁇ 2 H 2 O 0, 15 mg , H 3 BO 3 2, 5 mg, CoCl 2 ⁇ 6 H 2 O 0, 7 mg, CuSO 4 ⁇ 5 H 2 O 0.25 mg, MnCl 2 ⁇ 4 H 2 O 1.6 mg, ZnSO 4 ⁇ 7 H 2 O 0.3 mg, KH 2 PO 4 3.0 g, K 2 HPO 4 12.0 g, (NH 4 ) 2 SO 4 5 g, NaCl 0.6 g, FeSO 4 ⁇ 7 H 2 O 0.002 g, Na 3 citrate x 2 H 2 O 1 g, glucose 15 g, tryptone 1 g, yeast extract 0.5 g.
  • the SAM contained in the culture supernatant was quantified by HPLC.
  • a Develosil RP-aqueous C 30 column, 5 microns, 250 * 4.6 mm (Phenomenex, Aillesburg) was used and 10 .mu.l applied culture supernatant by isocratic elution with a flow agent of 3 ml of 85% H 3 PO 4 , on 1 1 H 2 O separated at a flow rate of 0.5 ml / min at room temperature and quantified by diode array detector at a wavelength of 260 nm.
  • Table 1 shows the achieved contents of SAM in the respective culture supernatant.
  • Table 1 tribe S-adenosylmethionine [mg / l] 24 hours 48 h W3110 0 0 W3110 / pMSRLSSk 79 63 W3110 / pFL274 287 222

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EP06003718A 2005-03-03 2006-02-23 Procédé pour la production de S-adénosylméthionine par fermentation Ceased EP1700864A1 (fr)

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EP2163613A3 (fr) * 2008-08-28 2013-01-02 Evonik Degussa GmbH Procédé de fabrication de liaisons organo-chimiques en utilisant des troncs améliorés de la famille des enterobacteriaceae

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US7851180B2 (en) * 2008-04-04 2010-12-14 Cj Cheiljedang Corporation Microorganism producing L-methionine precursor and the method of producing L-methionine precursor using the microorganism
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JP2014087259A (ja) 2011-02-22 2014-05-15 Ajinomoto Co Inc Ｌ−システイン生産菌及びｌ−システインの製造法
JP6020443B2 (ja) 2011-04-01 2016-11-02 味の素株式会社 Ｌ−システインの製造法
US9724132B2 (en) 2011-08-31 2017-08-08 DePuy Synthes Products, Inc. Devices and methods for cervical lateral fixation
US11053526B2 (en) 2018-08-09 2021-07-06 Evonik Operations Gmbh Process for preparing L amino acids using improved strains of the enterobacteriaceae family

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Cited By (3)

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Publication number Priority date Publication date Assignee Title
WO2007069782A1 (fr) * 2005-12-16 2007-06-21 Ajinomoto Co., Inc. Bacterie productrice d’acide l-amine et procede de production d’acide l-amine
US8008047B2 (en) 2005-12-16 2011-08-30 Ajinomoto Co., Inc. L-amino acid producing bacterium which has enhanced expression of at least one of the nhaA gene, the nhaB gene, the nhaR gene, the chaA gene, the mdfA gene and a method of producing L-amino acid
EP2163613A3 (fr) * 2008-08-28 2013-01-02 Evonik Degussa GmbH Procédé de fabrication de liaisons organo-chimiques en utilisant des troncs améliorés de la famille des enterobacteriaceae

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US20060211095A1 (en) 2006-09-21
US7410789B2 (en) 2008-08-12
CN1854303A (zh) 2006-11-01
JP2006238883A (ja) 2006-09-14
DE102005009751A1 (de) 2006-09-07

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